We have previously provided conclusive evidence that the invertebrate stages of Leishmania are fully capable of a sexual cycle during their growth and development in the sand fly vector. Our experimental mating attempts demonstrated mating compatibilities both within and between Leishmania species. Altogether, the outcrosses generated experimentally support the conclusion that the accumulating examples of hybrid genotypes observed in natural populations have likely arisen by genetic exchange. In addition to outcrosses, we have studied the self-mating competency in Leishmania, and have analyzed 26 self-mating progeny clones by whole genome sequencing and virulence phenotyping in mice. Results indicate that intraclonal mating is possible and can generate phenotypic diversity that is associated with a high frequency of genomic modifications, both polyploidy and aneuploidy. Since opportunities for outcrossing in the sand fly vector may be rare, self-mating may be the more important reproductive strategy underlying the remarkable diversity of the genus. Leishmania parasites have a dimorphic life cycle, shifting between the alimentary tract of their sand fly vector as extracellular, flagellated promastigotes, and the phagolysosomal vacuoles of their mammalian host cell macrophages as intracellular amastigotes. The ability of the parasite to adapt to these radically different environments has been the focus of a number of studies comparing the transcriptomes, proteomes and metabolomes of amastigotes and promastigotes during their transformation in vitro. There are a multitude of conditions in the vector, and a number of distinct promastigote developmental stages, that are likely adapted to microenvironments in the fly that are absent when Leishmania are axenically grown in vitro. We used RNA-sequencing to profile for the first time the transcriptomes of L. major during its transition from amastigotes to procyclic promastigotes in the sand fly midgut, and during its subsequent development in vivo as nectomonad promastigotes, and finally its differentiation to the infective, metacyclic promastigote stage. The biggest change in the mRNA profiles occurred in the first two days after the infective feed when the parasite transformed from amastigotes to procyclic promastigotes, with a total of 1212 differentially expressed genes (DEGs). Of the top 25 mRNAs that are down regulated as the parasites differentiate into procyclic promastigotes, the majority were the genes encoding the amastin surface proteins, extracellular proteases, and ABC transporters. mRNAs that are associated with sugar transport and metabolism were significantly up-regulated with the transition from amastigotes to procyclic promastigotes, while other sources of energy such as amino acids and ketone bodies were taken up and/or metabolized in the later developmental forms. Comparisons between the different in vivo promastigote developmental stages revealed that each stage expressed a number of unique surface proteins. The results also showed down regulation in nucleosome assembly and cell replication related genes at the nectomonad stage, with translation increasing again during metacyclogenesis and remaining elevated in amastigotes. Different stress responses were highly upregulated in the metacyclic stage. Visceral leishmaniasis (VL), which is endemic in the northeast Indian state of Bihar, is thought to have an anthroponotic transmission cycle as no mammalian host other than humans has ever been shown to harbor the etiologic agent, Leishmania donovani. However, which infected humans act as important reservoirs for transmission of L. donovani to the sand fly vector, Phlebotomus argentipes, remains unknown. The possibilities include active VL cases, clinically cured cases, patients with post-kala-azar dermal leishmaniasis (PKDL), and infected but asymptomatic individuals. As asymptomatic infections are far more frequent than those progressing to disease, it is especially important to determine the reservoir potential of asymptomatic cases. The problem is being addressed using two approaches: 1) The application of forensic DNA methods to identify the human source of an infected blood meal, and 2) xenodiagnostic studies of well-defined subject groups using live vector sand flies. Our findings established the feasibility of the forensic DNA method to directly trace the human source of an infected blood meal, with constraints imposed by the requirement that the flies be recovered for analysis within 24 hours of their infective feed. We found that the ability to obtain readable human DNA fingerprints from sand flies depended entirely on the size of the blood meal and the kinetics of its digestion. For the xenodiagnosis studies, the preparatory phase required two years to build an insectary, establish a robust, closed colony of P. argentipes from wild stock collected from the VL endemic zone in Bihar, and finally to certify the colony as Leishmania and phleovirus free for feeding on human volunteers. This work was completed in April, 2016 and the xenodiagnostic studies initiated in July, 2016. These studies will continue with accrual of over 100 volunteers from each exposure group during the next two years. We have also performed xenodiagnostic studies at the NIH using the hamster model of VL that reproduces the progressive form of human disease, to study the transmission dynamics of L. donovani to a colonized vector, Lutzomyia longipalpis. The transmission from the sick hamsters to the flies after the first feeding was unexpectedly low, with an average of only 16% of the blood fed flies acquiring infection. By contrast, there was a strong increase in L. donovani transmissibility after multiple exposures to sand fly bites (47%) that was due to a higher blood parasitemia, suggesting that there is a systemic response to sand fly bites in the host that promotes release of infected cells into the peripheral blood and that is exploited by the parasite to favor its transmissibility.